Summary

The telencephalon shows vast morphological variations among different
vertebrate groups. The transcription factor neurogenin1
(ngn1) controls neurogenesis in the mouse pallium and is also
expressed in the dorsal telencephalon of the evolutionary distant zebrafish.
The upstream regions of the zebrafish and mammalian ngn1 loci harbour
several stretches of conserved sequences. Here, we show that the upstream
region of zebrafish ngn1 is capable of faithfully recapitulating
endogenous expression in the zebrafish and mouse telencephalon. A single
conserved regulatory region is essential for dorsal telencephalic expression
in the zebrafish, and for expression in the dorsal pallium of the mouse.
However, a second conserved region that is inactive in the fish telencephalon
is necessary for expression in the lateral pallium of mouse embryos. This
regulatory region, which drives expression in the zebrafish diencephalon and
hindbrain, is dependent on Pax6 activity and binds recombinant Pax6
in vitro. Thus, the regulatory elements of ngn1 appear to be
conserved among vertebrates, with certain differences being incorporated in
the utilisation of these enhancers, for the acquisition of more advanced
features in amniotes. Our data provide evidence for the co-option of
regulatory regions as a mechanism of evolutionary diversification of
expression patterns, and suggest that an alteration in Pax6
expression was crucial in neocortex evolution.

Introduction

In vertebrates, neurogenesis is a complex process that controls the
position, number, connectivity and neurophysiological properties of thousands
of different neurones. It has become obvious in recent years that many
features of the molecular mechanisms and principles controlling neurogenesis
have been conserved throughout animal evolution. The neurogenic processes in
the peripheral nervous system of the fruit fly Drosophila
melanogaster served as important paradigms to unravel the regulatory
principles of vertebrate neurogenesis
(Bertrand et al., 2002;
Chitnis, 1999;
Hassan and Bellen, 2000). The
Drosophila peripheral nervous system is specified by a hierarchical
series of cell-fate decisions, in which prepattern genes first define regions,
the so-called proneural clusters, that express proneural basic
helix-loop-helix (bHLH) proteins and have the potential to develop into
neurones (Chitnis, 1999). In
the subsequent step of lateral inhibition that involves the Delta/Notch
signalling system, committed neural precursors are selected from these
proneural regions (Simpson,
1997). The regulatory events in the neural plate of lower
vertebrates bear a strong resemblance to these patterning mechanisms in the
imaginal discs of Drosophila. As in Drosophila, bHLH
transcription factors related to the proneural genes demarcate regions of
neuronal precursors, from which, by a Delta/Notch-mediated process, committed
neurones are selected (Blader et al.,
1997; Chitnis,
1999; Ma et al.,
1996).

Central, but less well understood questions concern how the spatial aspects
of neurogenesis are controlled in the vertebrate neural plate/tube and how
these mechanisms have been modified during vertebrate evolution. Neurogenins
(ngns), which belong to the bHLH family and which are most closely related to
the Drosophila bHLH factors Biparous and Atonal, demarcate the
regions of primary neurogenesis in the neural plate of zebrafish and
Xenopus embryos (Blader et al.,
1997; Ma et al.,
1996). Accumulating evidence suggests that these
ngn1-expressing regions in the neural plate of lower vertebrates are
specified by pre-pattern genes that provide positional codes with their large
and overlapping domains of expression
(Bally-Cuif and Hammerschmidt,
2003). Genes of the iroquois family, members of which are
involved in positioning the expression of the proneural genes achaete
and scute in the Drosophila imaginal disc, have been
suggested to play similar roles in the vertebrate neural plate
(Bellefroid et al., 1998;
Cheng et al.,2001;
Gomez-Skarmeta et al., 1998;
Itoh et al., 2002;
Wang et al., 2001). In
Xenopus embryos, the zinc-finger transcription factor Zic2 acts as a
repressor of ngn1 expression in the longitudinal stripes, separating
the ngn1-expressing precursors of motor neurones, interneurones and
sensory neurones (Brewster et al.,
1998). Another negatively acting factor, the bHLH factor Her5,
prevents neurogenesis at the midbrain/hindbrain boundary (MHB). Inhibition of
Her5 function leads to an expansion of ngn1 expression and ectopic
formation of a proneural field in the MHB area
(Geling et al., 2003).

In the neural tube of mouse embryos, the paired-homeodomain transcription
factor Pax6 has important regulatory functions
(Ashery-Padan and Gruss, 2001;
Simpson and Price, 2002). Pax6
mutant mice develop small eyes and have deficiency in neurogenesis in the
brain and spinal cord. In both the telencephalon and the spinal cord, Pax6 is
expressed in a graded fashion, suggesting that it provides
concentration-dependent positional information for the region-specific
differentiation of neural tissues
(Stoykova et al., 2000;
Scardigli et al., 2003). The
zebrafish genome encodes two pax6-related genes, pax6.1 and
pax6.2, that are expressed in overlapping domains in the eye, dorsal
diencephalon, hindbrain, spinal cord and pancreas, in a pattern reminiscent of
the pattern of pax6 expression in the mouse embryo
(MacDonald et al., 1994;
Nornes et al., 1998).
Interestingly, prominent expression of pax6.1 and pax6.2 was
not detected in the proliferating telencephalon of the zebrafish, in contrast
to mouse embryos (MacDonald et al.,
1994; Stoykova et al.,
2000; Wullimann and Rink,
2002). The pax6-expressing cells in the zebrafish
telencephalon constitute a small population of migrating, post-mitotic cells
at the pallial/subpallial border. By contrast, pax6 is abundantly
expressed in the proliferating radial glia cells of the mouse telencephalon.
This suggests that the pattern of pax6 expression was modified during
vertebrate brain evolution.

Expression of ngn2 in the mouse depends on pax6 activity
in both the spinal cord and the telencephalon
(Stoykova et al., 2000;
Scardigli et al., 2003). It
was recently shown that the ngn2 upstream region contains a
pax6-dependent regulatory region that drives expression in the spinal
cord (Scardigli et al., 2003).
Moreover, ngn2 expression in the telencephalon of the mouse depends
on pax6 activity (Stoykova et
al., 2000). Based on the expression of pax6.1 and
pax6.2 in the zebrafish neural plate/tube, pax6 may possess
equivalent functions in the control of the related ngn1 gene during
primary neurogenesis in the zebrafish embryo.

We have previously mapped the regulatory regions of ngn1
responsible for driving reporter expression in the neural plate. Two regions,
the lateral stripe element (LSE) and the anterior neural
plate element (ANPE) were identified in the ngn1
upstream region (Blader et al.,
2003). The LSE is required for expression in precursors
of Rohon Beard sensory neurones and reticulospinal neurones in the anlage of
the spinal cord and hindbrain, respectively; the ANPE is responsible
for expression in the ventral caudal cluster in the midbrain anlage, the
trigeminal ganglia and a few scattered nuclei in the anterior hindbrain
(Blader et al., 2003). Further
analysis of ANPE showed that it contained an E-box known to interact
with bHLH factors. Indeed, Her5 was demonstrated to regulate the activity of
ANPE, as in embryos that lacked Her5, expression of a transgene that
contained the ANPE was expanded into the MHB area
(Geling et al., 2004).
Moreover, mutation of the E-box in the ANPE caused an expansion of
reporter gene expression into the MHB area, suggesting that the E-box is
required for the suppression of transgene activity in the MHB by Her5
(Geling et al., 2004).

These cis-regulatory regions show homology with sequences at the mouse and
human ngn1 loci, despite the fact that mammals do not express
ngn1 in the neural plate, but only later in the neural tube
(Blader et al., 2003;
Simmons et al., 2001). This
suggests that these regions have shared functions in neurogenesis in mammals
and teleosts. The preliminary analysis of transgenes lacking the LSE
and ANPE in post-somitogenesis-stage zebrafish embryos suggested that
more proximal regions of the zebrafish ngn1 gene have regulatory
activity at later stages when the neural tube has formed
(Blader et al., 2003).

To delineate the regulatory regions responsible for brain expression of
ngn1 in older zebrafish embryos, we analysed transgenic lines
carrying wild-type and deletion variants of ngn1 transgenes. We
mapped two regulatory regions that are required for transgene expression in
the brain of post-somitogenesis-stage embryos. The first region, residing at
position –6702 to –6490 bp upstream of the ATG start site, which
was also previously shown to harbour the LSE, drives expression in
the dorsal telencephalon. A second regulatory region referred to as
LATE was mapped to position –1775 to –1368. The
LATE region, like the LSE, is highly conserved in mouse and
human homologues of ngn1. We carried out comparative functional
studies in mouse embryos to investigate the activity of these conserved
regulatory elements. We focused on the dorsal telencephalon of the mouse, as
this is undoubtedly the most derived brain region to have arisen during
vertebrate evolution (Nieuwenhuys,
1994; Wullimann and Rink,
2002). The LSE drives expression in the dorsal
telencephalon in both mouse and zebrafish embryos, indicating a conserved
function with respect to telencephalic expression. Curiously, we found that
the LATE region of the zebrafish ngn1 gene drives expression
in the lateral telencephalon of the mouse embryo but not in the zebrafish
telencephalon. The area of activity of LATE overlaps with that of the
paired-homeodomain transcription factor Pax6, suggesting a role of Pax6 in
regulating the activity of LATE. We demonstrate that Pax6 binds to a
conserved Pax6-binding site in the LATE region. Moreover, the lack of
pax6 activity in zebrafish by simultaneous knockdown of both
pax6.1 and pax6.2 leads to a small eye phenotype and
strongly reduces endogenous ngn1 and transgene expression. These
results are consistent with a direct regulatory role of Pax6 on the activity
of LATE. Based on the highly modular structure of vertebrate
regulatory regions, which are usually composed of multiple short and
degenerate binding sites for transcription factors, it is commonly assumed
that elaboration of novel patterns of gene expression is accomplished by
changes in the regulatory sequence
(Ludwig, 2002;
Stone and Wray, 2001;
Tautz, 2000). Our data suggest
that a pre-existing enhancer was co-opted, and that the evolution of the
pax6 expression pattern led to the recruitment of LATE into
the newly developed territories of the mouse telencephalon.

Materials and methods

Reporter constructs

The –8.4, –8.4(del1-9), –6.3,–
5.9 and –3.1 ngn1:gfp transgenes were published
previously (Blader et al.,
2003). Versions of all reporter constructs were also made using a
nuclear-localised β-galactosidase reporter. The delLATE
deletions were generated by a PCR strategy. Two PCR products 5′ and
3′ of the homology region were amplified using the oligonucleotide
pairs:
5′-TAATACCCGGGGATTAATGC-3′/5′-GATCGTCGACCACCCCGCTTCTGAGACACG-3′;
and
5′-CTGAGTCGACAATAAAACTTAAGCCACTGG-3′/5′-CTGTCCTGCATGCAACAAGC-3′.
They were ligated using SalI sites, which replace the homology region
between –1775 and –1368, and reamplified with the
5′-TAATACCCGGGGATTAATGC-3′/5′-CTGTCCTGCATGCAACAAGC-3′
oligonucleotide pair. The resulting PCR fragment was cloned into pBL3.1, by
replacing the XmaI/EcoRI fragment between –3122 and–
673. A 2.7-kb XmaI/NcoI fragment containing the
deletion [–3.1del(LATE)] was then cloned into a vector with the GFP:SV40
poly-A cassette. The 8.4(delLATE):GFP was made by introducing a 5283 bp
NotI/XmaI fragment containing the promoter-distal
ngn1 region (Blader et al.,
2003) in pBL-3.1del(LATE), and inserting the resulting–
8.4del(LATE) NotI/NcoI fragment into the vector with
the GFP:SV40 poly-A cassette. In the zebrafish/mouse chimeric constructs, the
regions of conservation (LSE, LATE) were replaced by the cognate
mouse sequence using standard PCR-based cloning strategies. Details on the
constructions are available upon request.

Transgenic animals and morpholino knockdown

Reporter fragments for generating transgenic fish were excised from
plasmids and separated by agarose gel electrophoresis, followed by
purification with the Qiaex II Kit (Qiagen), according to the manufacturer's
instructions. Fragments were diluted to 50 ng/μl in TE and injected into
freshly fertilised zebrafish embryos, as previously described
(Blader et al., 2003). Mouse
transgenics were generated as previously described
(Scardigli et al., 2001).
Morpholinos (GeneTools) were designed complementary to the 5′ region of
pax6.1 (5′-TTTGTATCCTCGCYGAAGTTCTTCG-3′) and
pax6.2 (5′-CTGAGCCCTTCCGAGCAAAACAGTG-3′) mRNA. They were
resuspended in 1×Danieau buffer [58 mM NaCl, 0.7 mM KCl, 0.4 mM
MgSO4, 0.6 mM Ca(NO3)2, 5.0 mM HEPES, pH 7.6]
and stored at 4°C. The morpholinos were injected into the yolk of
zebrafish embryos at the one- to two-cell stage, and at concentrations of
between 0.6 mM and 1.2 mM.

Electrophoretic mobility shift assays

Pax6 protein was produced in vitro using the Sp6TNT kit (Promega) according
to the manufacturer's protocol. Oligonucleotides containing the site C
sequence (5′-GGCTTTGATATATCATACATGCCTGAAGACTCCC-3′), or clusters
of point mutations (shown in bold)
(5′-GGCTTTGATAGCGACGCACGTAAGTCCGACTCCC-3′) were annealed by
heating to 90°C with an equimolar mixture of the upper and the lower
strands, and cooling slowly to room temperature. Annealed oligonucleotides
were labelled with [γ32ATP] using T4 polynucleotide kinase.
Binding reactions were performed in a total volume of 25 μl containing 10
mM Hepes (pH 7.9), 100 mM KCl, 4% Ficoll, 1 mM EDTA, 1 mM DTT and 2.5 μg of
poly(dI-dC). The reactions contained 4 μl of protein and 30,000 cpm of
probe. The reactions were allowed to proceed for 30 minutes at 4°C and
were analysed on a 6% polyacrylamide gel containing 0.25×Tris
borate-EDTA (TBE).

Results

To identify the regulatory elements for forebrain expression, transgenic
zebrafish that harbour the 8.4-kb sequence upstream of the coding region of
the zebrafish ngn1 locus fused to a green fluorescent
protein (gfp) reporter were analysed
(Blader et al., 1997;
Blader et al., 2003). GFP
expression was assayed at 28 hours post-fertilisation (hpf), a stage when the
forebrain is predominantly proliferative and expresses endogenous
ngn1 (Korzh et al.,
1998; Wullimann and Knipp,
2000). Four stable transgenic lines were analysed, whose embryos
displayed identical patterns of GFP expression with strong activity in the
telencephalon (Fig. 1A). In
general, endogenous ngn1 transcripts are abundantly expressed at this
stage and present a complex pattern of expression
(Fig. 1B). Besides the
telencephalon, ngn1 transcripts are also located in several clusters
within the diencephalon. At least six populations occupying distinct
dorsoventral regions can be recognised on whole-mounted embryos
(Fig. 1B). In the dorsal
diencephalon, ngn1 mRNA is detected in the epiphysis and the
pretectum. A larger, more prominent cluster in the dorsal thalamus, presumably
also comprising the future habenular nuclei, is located immediately adjacent
to the epiphysis. Further ventrally, ngn1 is found in the ventral
thalamus, the preoptic area and the posterior tuberculum. In the
mesencephalon, transcripts are localised predominantly in the tegmentum. With
the exception of the epiphysis, these domains of expression broadly agree with
the regions identified by Mueller and Wullimann
(Mueller and Wullimann, 2003)
based on the analysis of sections at 48 hpf. Comparison of the pattern of
expression of the endogenous ngn1 gene with that of the
–8.4ngn1:gfp transgene indicates that the domains demarcated by
GFP reproduced the pattern of endogenous ngn1 mRNA
(Fig. 1A,C). The expression of
the transgene is more intense than that of the endogenous gene. However, this
difference appears to be due to the higher copy number of the transgene, and
to the higher stability of the gfp reporter mRNA in comparison with
the endogenous ngn1 mRNA, which is expressed in a highly dynamic
pattern (Blader et al.,
1997).

The –8.4ngn1:gfp zebrafish transgene recapitulates the
pattern of endogenous ngn1 transcripts and drives telencephalic
expression in the zebrafish embryo. (A) The 8.4 kb upstream zebrafish
ngn1 regulatory sequence drives GFP expression in the telencephalon
(arrow) and in the diencephalon. (B,C) Comparison of the endogenous
ngn1 gene (B) and the gfp transgene (C) by in situ
hybridisation indicates that the gfp transgene is capable of
recapitulating the endogenous telencephalic (t, indicated by arrows) and
diencephalic expression of ngn1. Expression is detected in at least
six regions of the diencephalon, comprising the epiphysis (e), pretectum
(pre), dorsal thalamus (dt), ventral thalamus (vt), preoptic area (po) and
posterior tuberculum (pt). Transcripts are also localised in the midbrain
tegmentum (tg). Zebrafish embryos are at stage 28 hpf. Panels show lateral
views of whole-mounted embryos oriented anterior to the left and dorsal
up.

The regulatory region required for telencephalic expression maps to
the LSE

To identify sequences responsible for the activity of the
–8.4ngn1:gfp transgene in the zebrafish telencephalon, 5′
deletions retaining 6.9 kb (1 transgenic line), 5.3 kb (1 transgenic line) and
3.1 kb (2 transgenic lines) of the original 8.4 kb fragment upstream of the
GFP reporter (Blader et al.,
2003) were analysed. Telencephalic expression of GFP was lost in
the transgenic line containing 5.3 kb of upstream sequence, indicating that
the required regulatory elements lie between –6.9 and –5.3 kb
upstream of the ngn1 start codon
(Fig. 2A,B). The LSE,
which drives expression in the lateral neural plate, was previously mapped to
this region (Blader et al.,
2003). To assess whether the LSE and the region mediating
telencephalic expression co-localise, a series of eight overlapping 400-bp
deletions spanning this region [–8.4(del1-9)ngn1:gfp] was
analysed (Fig. 2C). The stable
lines carrying del2 (–6886 to –6490 nucleotides) and
del3 (–6702 to –6294 nucleotides) were found to lack
telencephalic GFP expression (Fig.
2C), identifying sequences between –6702 and –6490
nucleotides upstream of the ngn1 ATG as necessary for transgene
activity in the zebrafish telencephalon. The same region was shown to be
required for expression in the lateral neural plate, and was found to be
highly homologous (60%) to an upstream sequence of the murine and human
Ngn1 locus (Blader et al.,
2003).

Identification of the regulatory elements required for telencephalic
expression of the –8.4ngn1:gfp transgene in the zebrafish.
(A,B) Analysis of zebrafish embryos transgenic for truncated versions of the
–8.4ngn1:gfp reporter fragment. Transgene expression in the
telencephalon (arrows) is no longer detected in embryos carrying transgenes
with 5.3 kb of ngn1 sequence. (C) Overlapping deletions spanning the
region between 6.9 and 5.3 kb reveal a 200-bp element, the LSE, that
is required for telencephalic expression of the –8.4 ngn1:gfp
transgene. The heads of embryos in A are seen from the dorsal aspect, with the
telencephalon up, and are staged between 28 and 30 hpf. Boxes in B,
corresponding to the LSE (blue) and LATE (green), indicate
regions of homology between the zebrafish, mouse and human ngn1 loci.
Each construct was analysed in one to three transgenic lines. Note that
expression patterns from the overlapping deletions provided an independent
further confirmation of the results. Transgenic lines varied in the intensity
of reporter expression, but no significant variations in the expression
pattern due to integration site effects were observed.

The 8.4-kb zebrafish transgene is capable of recapitulating
ngn1 expression in the mouse telencephalon

A potential functional conservation of ngn1 regulation between
mammals and zebrafish was tested by generating transgenic mouse embryos with
the zebrafish –8.4ngn1:gfp transgene. At 12.5 days post-coitum
(dpc), at the onset of neurogenesis in the mouse telencephalon and at a stage
comparable to the 28-hpf zebrafish forebrain, a comparison of GFP expression
in transgenic embryos with embryos stained with a Ngn1-antisense
probe indicated that the zebrafish transgene drives reporter expression
comparable to endogenous Ngn1 in the telencephalon
(Fig. 3A-C). Furthermore, the
transgene reproduced the sharp ventral border, just ventral to the
corticostriatal angle, and the lateral-to-medial gradient of Ngn1
expression seen in the mouse at this stage
(Fig. 3B,C). In contrast to the
exclusive localisation of Ngn1 transcripts in the ventricular zone,
GFP activity was also detected in the pre-plate layer of postmitotic neurones,
probably because of the stability of the reporter protein
(Fig. 3C).

Recapitulating the Ngn1 expression pattern in the mouse requires
two zebrafish ngn1 gene regulatory regions. (A) The 8.4 kb sequence
upstream of zebrafish ngn1 drives reporter expression in the mouse
telencephalon (arrow). (B,C) Comparison of the expression of the transgene
with the expression of the endogenous Ngn1 gene in coronal sections
(dorsal up) shows that this fragment recapitulates fully the endogenous
telencephalic expression of Ngn1. The sharp ventral boundary of Ngn1
and transgene expression is indicated by a line. (D) Whereas the complete–
8.4ngn1:gfp transgene recapitulates the endogenous pattern of
Ngn1 (C), reporter activity is lost dorsally (arrow), but remains
laterally (arrowhead), in transgenic lines with the LSE deleted
[–8.4(del3)ngn1:gfp]. (E) Expression in the lateral
telencephalon (arrowhead) is driven by an element within 3.1 kb of the
ngn1 regulatory region (–3.1ngn1:nlacZ). (F) Deletion
of LATE [–3.1(del LATE)ngn1:nlacZ] abolishes the
lateral activity of the –3.1ngn1:nlacZ transgene. Thus, two
regulatory regions of the zebrafish transgene control the spatial expression
of ngn1 in the mouse pallium. (G) Summary of the activities of
LATE and LSE in the dorsal telencephalon of mouse. m, mantle
zone; v, ventricular zone. Mouse embryos are at 12.5 dpc. Two transgenic lines
were analysed per construct. With the expception of A, which is a lateral view
of a whole-mounted mouse embryo, panels show coronal sections through the
telencephalon, with dorsal up.

The LSE is not sufficient to drive the entire expression in
the mouse telencephalon

The conservation of the regulatory sequences in mammalian Ngn1
orthologues, together with the ability of the zebrafish transgene to
recapitulate the expression of the mouse ngn1 gene in the
telencephalon, suggests that zebrafish and mammals employ a similar regulatory
strategy to drive expression of Ngn1 in the telencephalon. To confirm
that expression of –8.4ngn1:gfp in the mouse telencephalon does
require the LSE, transgenic mouse embryos were generated carrying the
–8.4(del3)ngn1:gfp fragment. Expression of GFP was lost in
dorsal and medial regions of the telencephalon in
–8.4(del3)ngn1:gfp transgenics
(Fig. 3D), in comparison with
the –8.4ngn1:gfp line (Fig.
3C). Surprisingly, the transgene carrying the deletion retained
high levels of gene expression in the region of the pallial-subpallial border,
including the lateral telencephalon, prospective basolateral amygdalar complex
and claustrum-endopiriform nucleus. Hence, the LSE appears to
recapitulate only the dorsal-most expression of endogenous Ngn1 in
the mouse telencephalon, and additional regulatory elements are responsible
for the lateral domain of telencephalic expression.

Given that the complete pattern of endogenous Ngn1 expression can
be obtained with the –8.4ngn1:gfp zebrafish transgene in mouse,
regulatory sequences controlling lateral expression must be included in this
sequence that are apparently inactive in the zebrafish telencephalon. Sequence
comparison between the zebrafish and mammalian ngn1 loci revealed a
region of homology located between –1775 to –1368 kb upstream of
the zebrafish ngn1 start codon (called LATE; 61% homologous
to mouse Ngn1; Fig.
7A). Consistent with the hypothesis of LATE being
necessary for reporter expression in the lateral pallium of the mouse, E12.5
embryos transgenic for –3.1ngn1:nlacZ exhibited transgene
activity in the telencephalon in a pattern very similar to that of
–8.4(del3)ngn1:gfp transgenics
(Fig. 3D,E). Deletion of the
LATE region [–3.1(delLATE)ngn1:nlacZ] rendered the
transgene inactive in the telencephalon
(Fig. 3F), indicating that
LATE is indispensable for this activity in the mouse. Thus,
expression of the –8.4ngn1:gfp zebrafish transgene in the
telencephalon of mouse is a composite of the activities of two regulatory
regions, LSE and LATE
(Fig. 3G).

The LATE region is conserved between tetrapods and zebrafish. (A)
Sequence comparison of the LATE regulatory region of zebrafish
ngn1 with that of human, mouse, chicken and Xenopus
tropicalis. The aligned sequences comprise nucleotides –1775 and–
1368 in the zebrafish ngn1 genomic regulatory region. In each
column, the nucleotides are printed on a black background when they are all
identical, or on grey background when one base is present in more than half of
the sequences. Boxed regions indicate regions where the zebrafish sequence is
similar to the Pax6 consensus-binding site
(Epstein et al., 1994). Three
sites (A, B and C) were scored. (B) Comparison of the three putative
Pax6-binding sites with the consensus-binding sequence.

The LATE region is required for expression in the
diencephalon and hindbrain of zebrafish embryos

Transgenic zebrafish carrying the –3.1ngn1:gfp construct
express the reporter strongly in the diencephalon and in the hindbrain
(Fig. 4A). To test whether the
conserved LATE region controls these aspects of ngn1
expression, we generated stable transgenic zebrafish lines in which
LATE was removed by deletion of the sequence between –1775 and–
1368 upstream of the ATG. When LATE was abolished
[–3.1(delLATE)ngn1:gfp], expression was strongly reduced in the
diencephalon and the hindbrain (Fig.
4B). Reporter gene expression in the posterior spinal cord was
unaffected by the deletion of LATE (data not shown), indicating that
the reduction of transgene expression is due to the lack of LATE and
not to the integration site. Furthermore, when LATE was deleted from
the –8.4ngn1:gfp construct, a similar reduction of reporter
expression in the diencephalon was observed
(Fig. 4D). However, expression
in the telencephalon was not affected by the lack of LATE in the
–8.4ngn1 transgenes (compare
Fig. 4C,D). This confirms the
conclusion from the deletion series (Fig.
2C) that LATE does not mediate telencephalic expression
in the zebrafish embryo, and demonstrates that the LSE can drive
expression in the telencephalon in the absence of LATE.

The LATE region is required for expression in the diencephalon and
hindbrain of zebrafish embryos. (A) The –3.1ngn1:gfp transgene,
harbouring LATE, drives gfp expression extensively in the
diencephalon and in the hindbrain. This pattern is partially reminiscent of
the expression of the two zebrafish pax6 genes (see
Fig. 5D,E). (B) Upon deletion
of LATE in –3.1(delLATE)ngn1:gfp transgenic embryos,
expression of gfp mRNA in the diencephalon and in the hindbrain is
strongly reduced. (C,D) Deletion of LATE in the
–8.4ngn1:gfp transgenics reduced reporter expression in the
diencephalon (arrowhead) but not in the telencephalon (t). Thus, the activity
of LATE is required for transgene expression in the diencephalon.
Embryos were oriented with anterior directed towards the left and dorsal
up.

The activity of LATE depends on Pax 6

The paired homeodomain transcription factor Pax6 is required for expression
of the related Ngn2 in the lateral pallium of the mouse embryo
(Stoykova et al., 2000;
Toresson et al., 2000).
Moreover, the ventral boundary of Pax6 expression
(Stoykova et al., 2000)
coincides with the ventral boundary of LATE activity
(Fig. 5A,B). We tested whether
Pax6 is necessary for the activity of LATE in the murine
telencephalon by crossing the transgene into a Pax6 mutant background
(small eye) (Stoykova et al.,
2000). Lack of Pax6 activity abolishes expression of the
–3.1ngn1:nlacZ transgene in the lateral area of the
telencephalon (Fig. 5C) in the
same manner as deletion of LATE does
(Fig. 3F). Thus, Pax6 activity
is necessary for expression of the zebrafish transgene in the mouse.

LATE requires the activity of Pax6 in the mouse. (A) Schematic of
the expression domain of Pax6 in the telencephalon of the mouse
[reproduced, with permission, from Stoykova et al.
(Stoykova et al., 2000)]. (B)
Control embryo carrying the –3.1ngn1:nlacZ transgene. (C)
Pax6sey/sey embryo carrying the –3.1ngn1:nlacZ
transgene, showing the loss of reporter expression, indicating that
LATE activity depends on Pax6. Coronal sections through the
telencephalon with dorsal up. (D,E) Lateral view of the two zebrafish pax
genes, pax6.1 (D) and pax6.2 (E) shows overlapping
expression in the fore- and hindbrain (insets). Staining is detected mainly in
the dorsal diencephalon, reminiscent of the –3.1ngn1:gfp
transgene (see Fig. 4A). m,
mantle zone; v, ventricular zone.

The two zebrafish pax6 genes, pax6.1 and pax6.2
(Fig. 5D,E) are expressed in an
overlapping pattern in the diencephalon and the hindbrain
(MacDonald et al., 1994;
Nornes et al., 1998).
Strikingly, these two domains are highly similar to the territory of
LATE activity (Fig.
4A). Common to both anmiotes and anamiotes, Pax6 mRNA is
mainly detected in the eyes and in the alar plate of the forebrain (comprising
the pretectum, and the dorsal and ventral thalamus), and in the spinal cord.
However, in contrast to in mouse embryos, pax6.1 and pax6.2
are not significantly expressed in the telencephalon of zebrafish embryos
(MacDonald et al., 1994;
Nornes et al., 1998;
Wullimann and Rink, 2001). As
shown in Fig. 5D,E, only a
small domain of expression in the telencephalon, at the so-called
pallial-subpallial boundary, is detected using both pax6.1 and pax6.2
riboprobes.

To test whether LATE activity in the zebrafish embryo is also
dependent on Pax6, as observed in the mouse, we knocked down Pax6 activity by
injecting a cocktail of two antisense morpholinos complementary to
pax6.1 and pax6.2 mRNA. Phenotypically, 60% (n=152)
of the injected embryos showed a reduction in the size of their eyes
(Fig. 6A,B), when compared with
wild type. Expression of the endogenous ngn1 gene
(Fig. 6D) (22%, n=41),
as well as the –8.4ngn1:gfp transgene (28%, n=71,
Fig. 6F), was significantly
reduced in the diencephalon and hindbrain of morpholino-injected embryos.
Expression in the telencephalon was not affected, indicating that the
LSE is not dependent on Pax6 activity. The injection of either the
pax6.1 or the pax6.2 morpholino alone did not cause an
effect, suggesting that the two pax6 genes act redundantly. Moreover,
the lack of an effect of each morpholino by itself, even at high
concentrations (1.2 mM), is strongly suggestive of a specific interaction of
the morpholinos with the pax6.1 and pax6.2 mRNA.

Pax6 is required for ngn1 and transgene expression in the
diencephalon of the zebrafish embryo. (A-F) Control (A,C,E) and
morpholino-injected (B,D,F) embryos hybridised to ngn1 (A-D) and
gfp (E,F) antisense probes. Expression of the endogenous
ngn1 mRNA and the transgene (–8.4ngn1:gfp) is reduced
in the diencephalon (arrowheads) but not in the telencephalon (t). Embryos
were injected with a cocktail of morpholino oligonucleotides directed against
pax6.1 and pax6.2. Embryos are 28 hpf. (A,B) Dorsal views of
embryos with anterior oriented upwards; (C-F) lateral views. e, eye.

A conserved binding site in LATE interacts with Pax6 protein
in vitro

Our results indicate that LATE activity is dependent on Pax6 in
both the zebrafish and the mouse. Examination of the sequence of the
LATE region (Fig. 7A)
revealed three putative Pax6-binding sites
(Epstein et al., 1994),
site-A, -B and -C. Interestingly, Site-C is the most conserved between mouse,
human, chicken, Xenopus tropicalis and zebrafish ngn1.

To test whether the three Pax6-binding site homologies in the LATE
region can bind Pax6 protein, we performed electromobility-shift assays with
recombinant mouse Pax6 protein that was synthesised by in vitro translation
(Scardigli et al., 2003).
Oligonucleotides comprising either site-A, site-B or site-C
(Fig. 7B) were
32P-labelled and incubated with recombinant Pax6 protein in the
presence of unlabelled oligonucleotide containing the homologous Pax6-binding
site, or an oligonucleotide in which the putative Pax6-binding site was
mutated by a cluster of point mutations. As a positive control, we used a
previously described consensus Pax6-binding site
(Czerny et al., 1993). The
site-C oligonucleotide (sC) gave a strongly shifted band that was not reduced
by unspecific competitor with a mutated binding site (msC), but was totally
abolished by the presence of competitor oligonucleotide with an intact
Pax6-binding site homology (Fig.
8). Site-A and site-B did not yield retarded protein-DNA complexes
(data not shown), suggesting either that they do not interact with Pax6 or
that they bind to the protein very inefficiently.

Pax6 binds to site-C of the LATE region. Electromobility-shift
assays with recombinant mouse Pax6 protein. Radioactively labelled
oligonucleotide containing site-C of the LATE region was incubated
with Pax6 protein without competitor oligonucleotide, or with a 50-fold molar
excess of cold site-C oligonucleotide (sC), or with an oligonucleotide that
contained a cluster of point mutations (msC) in the Pax6-binding site homology
domain. The site-C competitor abolished the shifted band, whereas the complex
formation was not affected by the presence of the mutated oligonucleotide,
demonstrating that the interaction of Pax6 protein is dependent on an intact
Pax6-binding site. As a positive control, an oligonucletide (Control)
harbouring the Pax6-binding site described by Czerny et al.
(Czerny et al., 1993) was
used.

In summary, these results demonstrate that Pax6 can interact directly with
the LATE region. The observed effects in the Pax6-deficient embryos
are thus likely to be due to the failure of Pax6 to activate ngn1
expression through interaction with the LATE region.

In zebrafish embryos, the mouse LATE and LSE
regions have the same regulatory activities as their cognate zebrafish
enhancers

The zebrafish LATE region drives expression in the lateral
telencephalon of the mouse but not of the zebrafish. The high conservation of
LATE suggests that it may be the target of similar regulatory
principles that are used in different places in the forebrain of the zebrafish
and the mouse. Its dependence on Pax6 activity in both mouse and zebrafish is
in support of this notion. Hence, one prediction is that the mouse
LATE region, like the zebrafish LATE region should be active
in the zebrafish diencephalons, but should not drive expression in the
telencephalon.

To test this hypothesis, zebrafish embryos were injected with constructs,
in which the zebrafish LATE was replaced with the mouse element
(Fig. 9). The embryos were
analysed at 26 hours after injection. To overcome the moscaism of such
transient expression patterns, we used the SceI meganuclease protocol
(Thermes et al., 2002), and
collected, in addition, accumulative expression maps by overlaying the
expression pattern from many independently injected embryos
(Fig. 9A-F). The replacement of
zebrafish LATE with the conserved mouse LATE sequences
produced embryos in which the expression of GFP was restricted to the
diencephalon (Fig.
9C,C′), in a pattern similar to that of the zebrafish
LATE region (Fig.
9A,A′). This is reminiscent of the spatial activity of the–
3.1ngn:gfp stable transgenic lines
(Fig. 4A). In addition,
injected embryos do not show expression in the telencephalon
(Fig. 9C,C′). As seen in
the stable transgenic lines, deletion of LATE abolished diencephalic
expression almost completely (Fig.
9B,B′). Taken together, this suggests that mouse
LATE is functionally similar to zebrafish LATE when
introduced into the zebrafish embryo. Moreover, these findings suggest that
the regulatory principles controlling LATE in the zebrafish
diencephalon were co-opted in the evolution of the lateral telencephalon of
the mouse. There is, however, some variation in the diencephalic pattern
driven by the mouse and zebrafish LATE enhancers, indicating that
changes have occurred during evolution at the level of fine-tuning of the
expression pattern within the diencephalon.

The cis-regulatory elements of mouse LATE and LSE direct
GFP expression in the zebrafish diencephalon and the telencephalon,
respectively. The left panels show a cumulative map of the expression (blue
dots; n, number of embryos analysed) of the reporter and the right
panels are representative images of embryos injected with the construct
indicated in the right bottom corner. Reporter gene expression was revealed by
hybridising embryos to the antisense gfp probe. Blue and red boxes in
the schematic drawings indicate zebrafish and mouse enhancers, respectively.
(A-C) Embryos injected with –3.1ngn1:gfp, with mutant
derivatives without the zebrafish LATE [–3.1ngn1(delLATE):gfp],
or with a replacement with the mouse LATE
[–3.1ngn1(msLATE):gfp]. Mouse LATE drives expression
in the zebrafish diencephalon in a pattern very similar to zebrafish
LATE. Like its zebrafish homologue, mouse LATE does not
mediate expression in the zebrafish telencephalon (indicated by arrows). (D-F)
Embryos were injected with the –8.4ngn1:gfp transgenes, with
mutants without the LSE [–8.4ngn1(delLSE):gfp], or
with the mouse LSE in place of the homologous zebrafish sequence
[–8.4ngn1(msLSE):gfp]. As shown in stable expression
experiments, the absence of the LSE significantly reduced the
expression of the reporter in the telencephalon. The replacement of the
zebrafish LSE with the homologous mouse LSE restored
reporter expression in the telencephalon.

Next, we tested whether the mouse LSE would also be active in the
zebrafish embryo (Fig.
9D-F′). In contrast to LATE, the zebrafish
LSE drives expression in homologous structures, the dorsal
telencephalon, in mouse and zebrafish. Replacement of the zebrafish
LSE (–8.4ngn1:gfp) with the mouse LSE
[–8.4ngn1(msLSE):gfp] produced embryos with transgene
expression in the telencephalon (compare
Fig. 9D,D′ with
9F,F′). Transient
expression of constructs without the LSE only rarely drove expression
in the dorsal telencephalon (Fig.
9E,E′). As expected from the presence of LATE in
the parental transgene (–8.4ngn1:gfp), these derived constructs
also showed prominent expression in the diencephalon. Thus, the structure and
the function of the LSE from mouse and zebrafish ngn1 are
evolutionarily conserved.

Discussion

We previously mapped two enhancer regions that control ngn1
expression in the zebrafish neural plate
(Blader et al., 2003). Here, we
have characterised the regulatory elements that drive expression in the
embryonic brain at later post-somitogenesis stages. One of the previously
mapped regulatory regions, the LSE, is required for expression in the
zebrafish telencephalon. An additional, more proximally located region,
LATE, mediates expression in the diencephalon and hindbrain. Both
regions are conserved in mouse and human Ngn1 genes, and both regions
are active in the mouse and zebrafish brain, indicating that not only
structural but also functional aspects of the two regulatory regions have been
maintained during vertebrate evolution. Moreover, we provide evidence for a
role of Pax6 proteins as regulators of ngn1 expression in the
zebrafish that probably involves a direct interaction of Pax6 with
LATE.

Pax6 as a pre-pattern gene in the zebrafish brain

Several lines of evidence suggest that Pax6 is a regulator of
LATE. First, the pattern of expression of pax6 precedes and
subsequently overlaps with that of LATE activity in the dorsal
diencephalon and in the hindbrain. Moreover, LATE contains a
pax6-binding site that is conserved in mammalian homologues of
ngn1 and that binds recombinant Pax6 in vitro. The simultaneous
knockdown of pax6.1 and pax6.2 in the zebrafish embryo leads
to the reduction of ngn1 and transgene expression in the hindbrain
and diencephalon, in a manner very similar to that observed following the
deletion of LATE from transgenes. Finally, transgene expression in
the mouse depends on a functional pax6 gene.

Like the pre-pattern genes her5, iroquois1 and iroquois7
(Geling et al., 2003;
Itoh et al., 2002), the
zebrafish pax6 genes (Krauss et
al., 1991; Nornes et al.,
1998) are expressed in broad domains in the neural plate from
early stages onwards. However, not all cells within these broad domains
express the transgene or the endogenous ngn1 gene. Rather
ngn1 expression is restricted to distinct clusters of neurones that
are located in at least six regions of the diencephalon, including
non-pax6-expressing territories. In particular, the transcripts are
detected in the epiphysis, pretectum, dorsal thalamus, ventral thalamus,
preoptic area and the posterior tuberculum, in contrast to the expression of
pax6.1 and pax6.2, which is predominantly located in the
alar plate of the forebrain. This suggests that pax6 genes define a
broad domain of competence in the diencephalon, in which other factors
cooperate to specify the precise sub-region of neurogenesis. Hence,
pax6 could be regarded to act as a pre-pattern gene in the zebrafish
neural plate/tube, in a similar fashion to iroquois or her5
genes.

Humans and mice carrying a loss-of-function allele of Pax6 show a
severe haplo-insufficiency, causing aniridia and small eye phenotypes,
respectively (Engelkamp and van Heyningen,
1996). This seems to be in contrast to the situation in zebrafish
embryos, where reduction of pax6 activity by knockdown of an
individual pax6 gene did not cause a visible effect in the embryo.
This suggests that the two pax6 genes in the zebrafish can act
redundantly, at least during early embryonic stages. However, knockdown of
gene function by a morpholino approach can impair gene function only
transiently: defects obvious at later stages could thus not be scored.

Expression of the mouse Ngn2 gene is dependent on Pax6
activity in the telencephalon and in the spinal cord
(Stoykova et al., 2000;
Scardigli et al., 2003). Mouse
Ngn1 and Ngn2 are expressed in similar but not identical
patterns in the mouse nervous system, suggesting that the two Ngn
genes are regulated by related mechanisms in the mouse that are derived from a
common ancestral gene. However, despite the strong conservation of other
regulatory sequences, such as the ANPE
(Blader et al., 2003), the
zebrafish LATE enhancer sequence is not conserved in
Ngn2.

The recruitment of LATE in the mouse appears to be linked to
evolution of the telencephalon

The telencephalon is one region of the brain where molecular changes in
regulatory activity are likely to have occurred most extensively, given the
vast expansion and morphological variations of these forebrain structures
among different vertebrate groups
(Nieuwenhuys, 1994). These
differences are particularly striking in regions flanking the pallial (dorsal
telencephalon)-subpallial (ventral telencephalon) boundary, which plays a
pivotal role in the establishment of neuronal diversity, and in the reception
of diverse developmental signals from dorsal and ventral domains of the
telencephalon (Molnar and Butler,
2002). Despite the anatomical differences in telencephalon
structure, comparative gene expression studies suggest that development of the
forebrain follows a similar `Bauplan' in all vertebrates, raising the question
of how morphological differences evolved
(Fernandez et al., 1998;
Reiner, 2000;
Striedter, 1997;
Zerucha et al., 2000).
ngn1 is expressed in the dorsal telencephalon in both zebrafish and
mouse (Blader et al., 2003;
Fode et al., 2000), and thus
represents an interesting case to study the modification of cis-regulatory
elements during evolution of the telencephalon.

We demonstrate that expression of ngn1 transgenes in the dorsal
telencephalon of zebrafish embryos is dependent on the activity of one
regulatory region (LSE), whereas expression in the developing
isocortex of mouse requires the activity of two distinct regulatory regions
(LSE and LATE). The zebrafish regulatory sequences
faithfully recapitulate the endogenous pattern of Ngn1 expression in
the mouse, including the position of the sharp ventral boundary and the
high-lateral to low-medial gradient in the pallium. LATE is highly
conserved in the murine Ngn1 and Ngn2 genes
(Blader et al., 2003;
Scardigli et al., 2003). The
homologous region of Ngn2 is a direct target of Pax6 and
also drives expression in the lateral telencephalon
(Blader et al., 2003;
Scardigli et al., 2003). As
LATE is not required for lateral telencephalon expression in the
zebrafish, what maintained LATE over 450 million years of evolution?
Our data show that zebrafish LATE is employed to drive expression in
the diencephalon of the zebrafish, and that this function has also been
retained by the mouse LATE when placed into the context of the
zebrafish embryo.

Evolutionary modification of expression patterns

Eukaryotic regulatory regions are usually composed of multiple
protein-binding sites clustered within a few hundred base pairs or less. The
internal organisation of these regions can be rather flexible, as individual
protein-binding modules can vary in position and orientation, and the actual
DNA sequences bound by regulatory proteins are usually rather short and
degenerate. In addition, multiple regulatory regions that can be scattered
over megabases in vertebrate genomes contribute frequently to the expression
of a gene (Davidson et al.,
2000). Given this flexibility in the cis-regulatory organisation
of vertebrate genes, the strong conservation of the position and sequences of
the individual regulatory regions (LSE, ANPE, LATE) of the
ngn1 gene is remarkable. Regulatory regions of different genes of the
same species can change during evolution at varying speeds
(Davidson, 2001). The
conservation of regulatory regions over 450 million years of independent
evolution is not only restricted to ngn genes but also includes other
regulators of neurogenesis, such as delta-d
(Dornseifer et al., 1997),
sonic hedgehog (Müller et
al., 2002; Müller et al.,
1999) and pax6
(Kammandel et al., 1999),
which is suggestive of a strong selection pressure to maintain the structure
of the regulatory regions of some genes.

It is believed that the evolutionary diversification of the body plan was
driven to a large extent by changes in gene expression, rather than by the
emergence of novel regulatory proteins
(Dermitzakis and Clark, 2002;
Ludwig, 2002;
Stone and Wray, 2001;
Tautz, 2000). In principal,
three ways of how the cis-regulatory region evolved can be envisaged.
Cis-regulatory regions could have emerged de novo by the accidental clustering
of protein-binding modules in close proximity in non-coding sequences.
Alternatively, existing regulatory regions could have been modified by the
deletion or addition of protein-binding sites, giving novel patterns of
regulatory activity. As a third possibility, enhancer sequences could have
been co-opted. In this case, the expression of the interacting transcription
factors was altered, placing a pre-existing enhancer into a different spatial
and/or temporal regulatory context. In addition, rearrangements of the genome
may have played crucial roles in redistributing these novel regulatory
activities among genes.

Our findings for the ngn1 enhancers suggest a scenario in which a
regulatory sequence has been co-opted in order to drive expression in a novel
context. Pax6 is not widely expressed in the telencephalon of early
post-somitogenesis-stage zebrafish embryos. Only a few migratory post-mitotic
telencephalic cells express Pax6 in the zebrafish embryo
(Wullimann and Rink, 2002).
This is in striking contrast to the widespread expression of Pax6 in the
proliferative radial glia in the telencephalon of the mouse embryo
(Gotz et al., 1998;
Stoykova et al., 2000). This,
together with the dependence of LATE activity on Pax6, indicates that
evolutionary modulation of Pax6 expression could have been involved in the
recruitment of LATE for Ngn1 expression in the lateral
telencephalon of the mouse. Co-option of a regulatory sequence to drive
expression at the pallial/subpallial border and in immediately adjacent
structures in the mouse is particularly intriguing, as this region is believed
to be absent from the zebrafish telencephalon, and to have appeared first in
the amniote lineage as a major prerequisite for the emergence of the cortex in
mammals (Molnar and Butler,
2002). However, it is unlikely that Pax6 is the only factor
involved in this recruitment. Other regions of the embryo that express Pax6 do
not show expression of endogenous ngn1 or the
LATE-containing transgenes. Moreover, even in the small group of
cells that express pax6 in the zebrafish telencephalon, Pax6 is not
sufficient to activate ngn1 expression, indicating that cooperating
factors are also necessary. This is reflected in the extended regions of
conserved sequence flanking the Pax6-binding site in LATE that
presumably represent the phylogenetic footprints of other conserved
transcription factors (see Fig.
7). Nevertheless, our data, together with the appearance of
extensive Pax6 expression in the radial glial cells of the mammalian
neocortex, suggest an important role of Pax6 as one of the factors that have
recruited Ngn1 expression in the telencephalon of mammals.

Conclusion

Our results provide evidence that the co-option of pre-existing enhancers
is a mechanism to diversify regulatory patterns during evolution. These
results have further implications: the attempts to delineate fields of
evolutionary homology on the basis of shared gene expression can be
misleading, as expression territories may be composites of the activities of
distinct regulatory regions that have evolved independently, as demonstrated
here for the LSE and the LATE regions of ngn1.
Moreover, in comparative genomic approaches to identify regulatory regions by
sequence conservation, the regulatory function cannot be inferred from
conserved sequences, as regulatory regions may have been co-opted in distinct
processes during evolution.

Itoh, M., Kudoh, T., Dedekian, M., Kim, C. H. and Chitnis, A.
B. (2002). A role for iro1 and iro7 in the establishment of
an anteroposterior compartment of the ectoderm adjacent to the
midbrain-hindbrain boundary. Development129,2317
-2327.

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